In modern times, refrigeration and, particularly, freezing have become common and preferred means for storage of biological materials. While refrigeration preserves some important properties of the samples, others continue to deteriorate at a slow but significant rate. Frozen storage may arrest most of this deterioration, but the combination of freezing and thawing introduces other changes which destroy other important properties.
In the modern world, frozen foods have become a mainstay of the human diet. To ensure a high quality product, sufficient for the demanding consumer""s palate, frozen vegetables in particular, and frozen desserts, such as ice cream, have been the subject of extensive research by food processors. It is now known that recrystallization can have a substantial negative impact on the taste and texture of frozen foods. The advent of frost-free freezers has exacerbated this situation, which has been more traditionally associated with temperature fluctuations during transportation. After a relatively short period of time at other than sub-zero temperatures or even at sustained freezing temperatures, many frozen foods become less desirable, or worse, totally unsuitable, for human consumption.
While a variety of techniques have been implemented to mitigate the damages associated with recrystallization, and limited success has been attained, significant problems remain. Often, modifications to the processing of the frozen foods drastically affect their quality, color, flavor, and/or texture. Moreover, the additional processing can be very expensive and time consuming, rendering the techniques uneconomical. Similar difficulties have been associated with incorporating additives to the foodstuffs.
For biologics, such as therapeutic drugs, blood plasma, mammalian cells for use in tissue culture, and the like, freezing can cause extensive damage. For example, the freezing process itself kills most eukaryotic cells, and cells subjected to even one freezing and thawing cycle exhibit greatly reduced viability. Impaired function of living cells is also prevalent in tissue cryopreservation, with concomitant drawbacks for organ transplants. Similarly, frost or other freezing damage to plants presents a serious problem in agriculture. Finally, drugs can become ineffective, or even dangerous, if not maintained under required strict temperature conditions.
Although the first description of protein-mediated thermal hysteresis (TH) was noted in Tenebrio molitor approximately 30 years ago (Grimstone, et al., Philos. Trans. B 253:343 (1968)), numerous attempts to purify these thermal hysteresis proteins (THP) failed to yield pure fractions with enough TH to account for the hemolymph activity (Grimstone, et al., (1968); Patterson and Duman, J. Exp. Zool. 210:361 (1979); Schneppenheim and Theede Comp. Biochem. Physiol. 67B:561 (1980); Tomchaney, et al., Biochemistry 21:716 (1982); Paterson Duman J. Exp. Zool. 219:381 (1982); and (Horwath, et al., Eur. J. Entomol. 93: 419 (1996)). Homogeneity of these proteins was not proven, and they differed in amino acid composition from each other and from the compositions reported here.
There exists a need for new techniques and compositions suitable for improving the preservation characteristics of organic materials at low temperatures, including storage of frozen foods and the viability of biologics. Ideally, these techniques and compositions will be inexpensive, yet completely safe and suitable for human consumption or in vivo therapeutic uses. There also exists a need for new techniques and compositions suitable for depressing the freezing point or inhibiting freezing in non-organic systems, such as in deicing treatments. The present invention fulfills these and other needs.
The eastern spruce budworm, Choristoneura fumiferana, and western spruce budworm, Choristoneura occidentalis, are freeze-tolerant pests of North American forests. A defense against freezing is the thermal hysteresis (TH) activity of their hemolymph, which allows the insects to depress their freezing points in the presence of ice or ice nucleators. This activity is quantified as the temperature difference (xc2x0C.) between the freezing and melting points of a solution containing ice. Hemolymph from spruce budworm larvae demonstrates a freezing point depression of greater than 4xc2x0 C.
This invention provides for the nucleic acid molecules that encode the proteins responsible for the thermal hysteresis in Choristoneura larvae, such as the nucleic acid of SEQ ID NO:1. The invention also provides for an isolated nucleic acid encoding an antifreeze protein where the protein can be defined as follows: having a calculated molecular weight of between 7 and 15 kDa; having a thermal hysteresis activity greater than about 1.5xc2x0 C. at a concentration of about 1 mg/mL; and, specifically binding to an antibody raised against antifreeze proteins or antigenic fragments thereof selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3, or, having at least 60% amino acid sequence identity to an antifreeze protein selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3. In a preferred embodiment, the isolated nucleic acid encodes a protein with a calculated molecular weight between about 8 and 12 kDa. In a preferred embodiment, the nucleic acid encodes a protein with a thermal hysteresis activity that is greater than 2xc2x0 C. at a concentration of about 1 mg/mL. In a preferred embodiment, the nucleic acid encodes a protein with at least 80% sequence identity to an antifreeze protein selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3. In a further embodiment, the invention provides for a nucleic acid encoding a protein with the sequence listed in SEQ ID NO:2 and SEQ ID NO:3. In another embodiment, the isolated nucleic acid encodes an antifreeze protein found in insects, and in a preferred embodiment that insect is a Choristoneura sp. The invention also provides for an isolated nucleic acid which specifically hybridizes to SEQ ID NO:1 under stringent conditions. The invention further provides for an isolated nucleic acid from a purified Choristoneura sp. antifreeze protein which specifically binds to an antibody directed against antifreeze proteins selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3.
The invention provides for THP proteins, including the recombinant proteins derived from the nucleic acids of this invention. The THP precursor protein (SEQ ID NO:2) has an N-terminal 18 residue signal sequence (SEQ ID NO:11) which is cleaved to generate mature THP protein (SEQ ID NO:3), see FIG. 2, which indicates the cDNA (SEQ ID NO:17) and corresponding amino acid sequence of spruce budworm THP.
The invention also provides for antibodies raised against the proteins of this invention and antibodies that bind to the proteins of this invention. The invention provides for antibodies specifically imnmunoreactive under immunologically reactive conditions to an antifreeze protein comprising SEQ ID NO:2 and SEQ ID NO:3. The invention also provides for an antibody, specifically immunoreactive under immunologically reactive conditions, to an antifreeze protein comprising the protein encoded by the nucleic acid of claim 1.
In a further embodiment of this invention, transformed yeast, bacteria and transgenic organisms are provided for. Many frozen foodstuffs suffer from formation of ice crystals due to sustained subfreezing temperatures or repeated freeze-thaw cycles. The presence of the THP of this invention will provide for longer shelf-life make these foodstuffs more palatable. Transgenic animals and plants are envisioned as better surviving sub-freezing temperatures. The invention provides for an organism into which an exogenous nucleic acid sequence which specifically hybridizes under stringent conditions to SEQ ID NO:1 or the nucleic acid of claim 1 has been introduced, and the organism translates the exogenous nucleic acid into an antifreeze protein. Also provided for is an organism with an exogenous nucleic acid sequence which is translated into an antifreeze protein that is expressed externally from the organism. In a preferred embodiment, the organism is a fish. In further preferred embodiments, organism is a fish is kept in a salt-water environment, or, the fish is a member of the family Salmonidae. In other preferred embodiments, the organism can be a plant, a fungus, a yeast or a bacteria. In another embodiment, if the organism is a yeast, it can be selected from the group consisting of Torulopsis holmil, Saccharomyces fragilis, Saccharomyces cerevisiae, Saccharomyces lactis, and Candida pseudotropicalis. In another embodiment, if the organism is a bacterium, it can be selected from the group consisting of Escherichia coli, Streptococcus cremoris, Streptococcus lactis, Streptococcus thermophilus, Leuconostoc citrovorum, Leuconostoc mesenteroides, Lactobacillus acidophilus, Lactobacillus lactis, Bifidobacterium bifidum, Bifidobacteriu breve, and Bifidobacterium longum. 
The invention provides for a method for decreasing the freezing point of an aqueous solution involving the addition of an antifreeze protein to the aqueous solution. In a preferred embodiment, the method involves the addition of the antifreeze protein encoded by the nucleic acid of claim 1 to the aqueous solution. In other preferred embodiments, the aqueous solution is applied to an organism; the antifreeze protein is produced by recombinant means; the antifreeze protein can specifically bind to the antibody of claim 13 or claim 14; the antifreeze protein is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3; and, the antifreeze protein is encoded by a nucleic acid molecule which specifically hybridizes to the nucleic acid of SEQ ID NO:1.
In addition, it is contemplated that the addition of the THP of this invention to aqueous solutions may better preserve organs and other biologicals in transit.
There also exists a need for new techniques and compositions suitable for depressing the freezing point or inhibiting freezing in non-organic systems, such as in deicing treatments.
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification, the figures and claims.